Floid cells imaging station microscope

The assessment of ΔΨm was performed according to Ref. [95 (link)]. Briefly, cells were incubated for 20 min at RT in the dark, with a deep-red MitoTracker (20 nM final concentration) compound (cat #M22426, Thermo Scientific, Waltham, MA, USA). Cells were analyzed using fluorescence microscopy Floid Cells Imaging Station microscope (cat# 4471136, Life Technologies, Carlsbad, CA, USA), or a BD LSRFortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was conducted 3 times, and 10,000 events were acquired for analysis. MitoTracker highly positive cells were selected located between 10⁴ and 10⁶. No discrimination by complexity was made. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software (BD Biosciences, Franklin Lakes, NJ, USA).

To analyze lysosomal complexity, cells were incubated with the cell-permeable, non-fixable, green, fluorescent dye LysoTracker Green DND-26 (50 nM, cat #L7526, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 37 °C. Cells were then washed, and LysoTracker fluorescence was determined by analysis of fluorescence microscopy images in a Floid Cells Imaging Station microscope (Cat# 4471136, Life Technologies, Carlsbad, CA, USA), or flow cytometry using a BD LSRFortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was conducted 3 times, and 10,000 events were acquired for analysis. Flow cytometry analysis for LysoTracker/SSCA was performed by selecting, in the FL-1 channel, all cells with LysoTracker reactivity (>99%), in order to perform the analysis of the total LysoTracker-positive population. SSCA parameter was adjusted to the mean fluorescence of the control (UNT; 40 K ± 3.5 K) plus two standard deviations (i.e., values above 47 K). Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software (BD Biosciences, Franklin Lakes, NJ, USA).

Light microscopy photographs were taken using a Zeiss Axiostart 50 Fluorescence Microscope (Carl Zeiss, Gottingen, Germany) equipped with a Canon PowerShot G5 digital camera (Zeiss Wöhlk-Contact-Linsen, Gmb Schcönkirchen, Gottingen, Germany), and fluorescence microscopy photographs were taken using a Zeiss Axiovert A1 Fluorescence Microscope equipped with a Zeiss AxioCam Cm1 and (Zeiss Wöhlk-Contact-Linsfluoreen, Gmb Schcönkirchen, Gottingen, Germany) and Floid Cells Imaging Station microscope (Cat# 4471136, Life Technologies, Carlsbad, CA, USA). Fluorescence images were analyzed by ImageJ software (http://imagej.nih.gov/ij/, accesed on 7 May 2023). Mean fluorescence intensity (MFI) was obtained by normalizing total fluorescence to the number of nuclei.